Combination therapy

ABSTRACT

The present invention relates to a combination therapy of propane-1-sulfonic acid {3-[5-(4-chloro-phenyl)-1H-pyrrolo [2,3-b] pyridine-3-carbonyl-2,4-difluoro-phenyl]-amide}, or a pharmaceutically acceptable salt thereof, and an interferon for treating a patient suffering from a proliferative disorder, in particular a solid tumor, for example, colorectal cancer, melanoma, and thyroid cancer. In particular, the present invention relates to such a therapy wherein the interferon is peginterferon alfa-2a and the disorder is melanoma containing the V600E b-Raf mutation.

PRIORITY TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/422,690, filed Dec. 14, 2010, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a combination therapy for treating apatient suffering from a proliferative disorder, in particular a solidtumor, for example, colorectal cancer, melanoma, and thyroid cancer,comprising administering to the patient propane-1-sulfonic acid{3-[5-(4-chloro-phenyl)-1H-pyrrolo [2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl}-amide and an interferon.

BACKGROUND OF THE INVENTION

Normally functioning b-Raf is a kinase which is involved in the relay ofsignals from the cell membrane to the nucleus and is active only when itis needed to relay such signals. Mutant b-Raf containing a V600Emutation, however, is constantly active and thus plays a role in tumordevelopment. Such mutant b-Raf has been implicated in various tumors,for example, colorectal cancer, melanoma, and thyroid cancer.

Propane-1-sulfonic acid {3-[5-(4-chloro-phenyl)-1H-pyrrolo [2,3-b]pyridine-3-carbonyl]-2,4-difluoro-phenyl}-amide (hereafter also referredto as “Compound I”) is a b-raf kinase inhibitor that specificallytargets mutant b-Raf containing the V600E mutation. This compound isdescribed in WO 2007/002325. Accordingly, such an inhibitor is used inthe inhibition of tumors, particularly solid tumors, for example,colorectal cancer, melanoma, and thyroid cancer, which comprise b-Rafhaving the V600E mutation.

Interferons (IFNs) are naturally occurring proteins that have antiviral,antiproliferative, and immunoregulatory activity. The IFNα familyrepresents the predominant class of IFNs produced by stimulatedperipheral blood leukocytes and lymphoblastoid and myeloblastoid celllines. Interferons downregulate the expression of bFGF. These drugs canbe self administered by patients via subcutaneous injection withresultant good pharmacokinetics. For the purpose of the presentspecification, the term “interferon” shall refer also to modifiedinterferons and/or recombinantly produced interferons such aspeginterferon alfa-2a.

Peginterferon alfa-2a (sold as Pegasys® by Genentech, South SanFrancisco, USA) is a covalent conjugate of recombinant alfa-2ainterferon (having an approximate molecular weight of 20,000 daltons)with a single branched bis-monomethoxy polyethylene glycol (PEG) chain(having an approximate molecular weight of 40,000 daltons). The PEGmoiety is linked at a single site to the interferon alfa moiety via astable amide bond to lysine. Peginterferon alfa-2a has an approximatemolecular weight of 60,000 daltons. The advantage of peginterferonalfa-2a over interferons that do not contain the PEG moiety is thatpeginterferon alfa-2a exhibits a longer half-life, requiring lessfrequent dosing.

SUMMARY OF THE INVENTION

The present invention relates to a method of treating a patientsuffering from a proliferative disorder, comprising administering to thepatient: (A) a first component which comprises, as an active agent,Compound I, or a pharmaceutically-acceptable salt thereof; and (B) asecond component which comprises, as an active agent, an interferon; theamounts of said active agents being such that the combination thereof istherapeutically-effective in the treatment of said proliferativedisorder.

The present invention also relates to a kit comprising: (A) a firstcomponent which comprises, as an active agent, Compound I, or apharmaceutically-acceptable salt thereof; and (B) a second componentwhich comprises, as an active agent, an interferon.

The present invention further relates to a composition comprising: (A) afirst component which comprises, as an active agent, Compound I, or apharmaceutically-acceptable salt thereof; and (B) a second componentwhich comprises, as an active agent, an interferon.

In addition, the present invention relates to the use of Compound I, ora pharmaceutically-acceptable salt thereof, and an interferon for thetreatment of a proliferative disorder.

A yet further aspect of the present invention is the use of Compound I,or a pharmaceutically-acceptable salt thereof, and an interferon for thepreparation of a medicament for the treatment of a proliferativedisorder.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the tolerability, as demonstrated by % body weightchange, of Compound I 75 mg/kg bid monotherapy, peginterferon alfa-2a900 μg 1×/wk monotherapy, and Compound I 75 mg/kg bid/peginterferonalfa-2a 900 μg 1×/wk combination therapy.

FIG. 2 illustrates the antitumor activity, as demonstrated by tumorvolume, of Compound I 75 mg/kg bid monotherapy, peginterferon alfa-2a900 μg 1×/wk monotherapy, and Compound I 75 mg/kg bid/peginterferonalfa-2a 900 μg 1×/wk combination therapy.

FIG. 3 illustrates the effect on survival, as demonstrated by percentageof surviving mice over time, of Compound I 75 mg/kg bid monotherapy,peginterferon alfa-2a 900 μg 1×/wk monotherapy, and Compound I 75 mg/kgbid/peginterferon alfa-2a 900 μg 1×/wk combination therapy.

FIG. 4 illustrates the tolerability, as demonstrated by % body weightchange, of Compound I 25 mg/kg bid monotherapy, peginterferon alfa-2a450 μg 1×/wk monotherapy, and Compound I 25 mg/kg bid/peginterferonalfa-2a 450 μg 1×/wk combination therapy.

FIG. 5 illustrates the antitumor activity, as demonstrated by tumorvolume, of Compound I 25 mg/kg bid monotherapy, peginterferon alfa-2a450 μg 1×/wk monotherapy, and Compound I 25 mg/kg bid/peginterferonalfa-2a 450 μg 1×/wk combination therapy.

FIG. 6 illustrates the effect on survival, as demonstrated by percentageof surviving mice over time, of Compound I 25 mg/kg bid monotherapy,peginterferon alfa-2a 450 μg 1×/wk monotherapy, and Compound I 25 mg/kgbid/peginterferon alfa-2a 450 μg 1×/wk combination therapy.

DETAILED DESCRIPTION OF THE INVENTION

As stated above, “Compound I” shall herein refer to propane-1-sulfonicacid {3-[5-(4-chloro-phenyl)-1H-pyrrolo [2,3-b]pyridine-3-carbonyl-2,4-difluoro-phenyl]-amide}. This is a compoundhaving the following structure.

Compound I is a b-Raf kinase inhibitor that specifically targets theV600E mutation of b-Raf.

The “V600E” mutation of b-Raf, as used herein, refers to a mutation inthe b-Raf protein wherein the valine residue at residue position 600 ofb-Raf is replaced by glutamic acid.

As used herein, the term “pharmaceutically acceptable carrier” indicatesthat the indicated carrier does not have properties that would cause areasonably prudent medical practitioner to avoid administration thereofto a patient, taking into consideration the disease or conditions to betreated and the respective route of administration.

As used herein, the term “pharmaceutically acceptable salt” of acompound refers to any conventional salt or base addition salt thatretains the biological effectiveness and properties of the compound andwhich is formed from a suitable non-toxic organic or inorganic acid ororganic or inorganic base.

As used herein, the term “therapeutically effective” means an amount ofdrug, or combination or composition, which is effective for producing adesired therapeutic effect upon administration to a patient, forexample, to stem the growth, or result in the shrinkage, of a canceroustumor or to increase the patient's life span.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the proliferative disorder is cancer.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include, butare not limited to, colorectal cancer, melanoma, and thyroid cancer.

The term “colorectal tumor” or “colorectal cancer” refers to any tumoror cancer of the large bowel, which includes the colon (the largeintestine from the cecum to the rectum) and the rectum, including, e.g.,adenocarcinomas and less prevalent forms, such as lymphomas and squamouscell carcinomas.

“Inhibiting cell growth or proliferation” means decreasing a cell'sgrowth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or 100%, and includes inducing cell death.

The phrase “substantially reduced” or “substantially different,” as usedherein, refers to a sufficiently high degree of difference between twonumeric values (generally one associated with a molecule and the otherassociated with a reference/comparator molecule) such that one of skillin the art would consider the difference between the two values to be ofstatistical significance within the context of the biologicalcharacteristic measured by said values.

The term “tumor” refers to all neoplastic cell growth and proliferation,whether malignant or benign, and all pre-cancerous and cancerous cellsand tissues. The terms “cancer,” “cancerous,” “cell proliferativedisorder,” “proliferative disorder,” and “tumor” are not mutuallyexclusive as referred to herein.

“Regression” of a tumor is said to occur following treatment when thevolume of said tumor is reduced. If the tumor remains present (tumorvolume >0 mm³) but its volume is reduced from what it was at theinitiation of treatment, “partial regression” (PR) is said to haveoccurred. If the tumor is palpably absent following treatment, “completeregression” (CR) is said to have occurred.

The present invention relates to a method of treating a patientsuffering from a proliferative disorder, comprising administering to thepatient: (A) a first component which comprises, as an active agent,Compound I, or a pharmaceutically-acceptable salt thereof; and (B) asecond component which comprises, as an active agent, an interferon; theamount of said active agents being such that the combination thereof istherapeutically-effective in the treatment of said proliferativedisorder.

Treatment of a proliferative disorder shall be understood to includemaintaining or decreasing tumor size, inducing tumor regression (eitherpartial or complete), inhibiting tumor growth, and/or increasing thelife span of a patient suffering from said disorder.

The present invention also relates to a kit or a composition comprising:(A) a first component which comprises, as an active agent, Compound I,or a pharmaceutically-acceptable salt thereof; and (B) a secondcomponent which comprises, as an active agent, an interferon. The kit orcomposition may be used, for example, in the treatment of a poliferativedisorder.

In addition, the present invention provides the use of Compound I, or apharmaceutically-acceptable salt thereof, and an interferon for thetreatment of a proliferative disorder.

The invention further provides the use of Compound I, or apharmaceutically-acceptable salt thereof, and an interferon for thepreparation of a medicament for the treatment of a proliferativedisorder.

In an embodiment of the present invention, the patient is a human.

In an embodiment of the invention, the proliferative disorder is a solidtumor.

In another embodiment of the invention, the proliferative disorder is atumor containing the V600E b-Raf mutation.

In a further embodiment of the invention, the tumor is a solid tumor isselected from the group consisting of: colorectal cancer, melanoma, andthyroid cancer and the cancer involves a tumor comprising b-Raf havingthe V600E mutation.

In yet a further embodiment of the invention, the proliferative disorderis a solid tumor comprising b-Raf having the V600E mutation.

In yet a further embodiment of the invention, the tumor is a solid tumorcontaining the V600E b-Raf mutation and said tumor is selected from thegroup consisting of: colorectal cancer, melanoma, and thyroid cancer.

In yet a further embodiment of the invention, the tumor is melanoma.

In yet a further embodiment of the invention, the tumor is melanomacontaining the V600E b-Raf mutation.

In yet a further embodiment of the invention, the interferon is selectedfrom the group consisting of: peginterferon alfa-2a, interferon alfa-2a,peginterferon alfa-2b, and interferon alfa-2b.

In yet a further embodiment of the invention, the interferon ispeginterferon alfa-2a.

In yet a further embodiment of the invention, the interferon isinterferon alfa-2b.

In yet a further embodiment of the invention, the present inventionrelates to a method of treating a patient suffering from melanomacontaining the V600E b-Raf mutation, comprising administering to thepatient: (A) a first component which comprises, as an active agent,Compound I, or a pharmaceutically-acceptable salt thereof, and (B) asecond component which comprises, as an active agent, peginterferonalfa-2a; the amount of said active agents being such that thecombination thereof is therapeutically-effective in the treatment ofsaid melanoma.

The amount of each component administered according to the presentmethod may, but does not have to be therapeutically effective by itself.That is, this invention specifically contemplates combinations whereinthe amount of Compound I, or a pharmaceutically-acceptable salt thereof,and/or the amount of interferon, in the combination may be less than theamount that is therapeutically-effective for each active agent when saidagent is administered in monotherapy.

Compound I, or a pharmaceutically-acceptable salt thereof, may, forexample, be administered orally. Peginterferon alfa-2a may, for example,be administered subcutaneously. The first component and the secondcomponent of the present invention are administered in any amount andfor any duration that the combined amounts thereof are therapeuticallyeffective in treating a proliferative disorder.

In embodiments of the present invention, Compound I is administereddaily at a dosage amount of from about 200 mg/day to about 3000 mg/day,from about 800 mg/day to about 2500 mg/day, from about 1400 mg/day toabout 2100 mg/day, about 960 mg/day, about 1440 mg/day, or about 1920mg/day.

In an embodiment of the present invention, the foregoing amounts ofCompound I may be administered as a single dose daily or divided, forexample into equal doses (though this is not required), and administeredtwice daily (bid). For example, Compound I may be administered daily ina dosage amount of from about 100 mg to about 1500 mg bid, from about400 mg to about 1250 mg bid, from about 700 mg to about 1050 mg bid,about 480 mg bid, about 720 mg bid, or about 960 mg bid.

In an embodiment of the present invention, the administration ofCompound I, or a pharmaceutically acceptable salt thereof, occurs untildisease progression or unacceptable toxicity.

In an embodiment of the present invention, peginterferon alfa-2a isadministered at a dosage of from about 1 μg/week to about 1,000 μg/week,from about 50 μg/week to about 800 g/week, or from about 90 μg/week toabout 630 μg/week. In yet another embodiment, the dosage amount is about180 μg/week.

In an embodiment of the present invention, the administration ofpeginterferon alfa-2a occurs until disease relapse, disease progressionor unacceptable toxicity. In another embodiment, the peginterferonalfa-2a is administered at the dosage amounts described above for aperiod of up to 12 months, up to 24 months, up to 36 months, or up to 60months.

The present invention also provides a method of treating a patientsuffering from a proliferative disorder, comprising administering to thepatient: (A) a first component which comprises, as an active agent,Compound I in an amount of from about 200 mg/day to about 3000 mg/day,from about 800 mg/day to about 2500 mg/day, from about 1400 mg/day toabout 2100 mg/day, about 960 mg/day, about 1440 mg/day, or about 1920mg/day; and (B) a second component which comprises, as an active agent,peginterferon alfa-2a in an amount of from about 1 μg/week to about1,000 μg/week, from about 50 μg/week to about 800 μg/week, from about 90μg/week to about 630 g/week, or about 180 μg/week. In an embodiment ofthis invention, the proliferative disorder is a solid tumor, inparticular the disorder is selected from the group consisting of:colorectal cancer, melanoma, and thyroid cancer. In another embodimentof this invention, the proliferative disorder involves a tumorcomprising b-Raf having the V600E mutation. In a particular embodimentof this invention, the proliferative disorder is melanoma comprisingb-Raf having the V600E mutation.

In another aspect of this invention, the components herein describedabove are administered in conjunction with radiotherapy and/or inconjunction with another active agent.

Compound I exists in its natural state in a crystalline form. However,the amorphous form of the compound has greater solubility in water ascompared with the crystalline form and thus has an improved dissolutionrate and, therefore, improved bioavailability as compared to thecrystalline form. As such, the amorphous form of the compound ispreferred. Accordingly, in preferred embodiments of the presentinvention, Compound I is in substantially amorphous form and, morepreferably, in amorphous form. As used herein, the term “substantiallyamorphous” material embraces material which has no more than about 10%crystallinity; and “amorphous” material embraces material which has nomore than about 2% crystallinity.

In an embodiment of the present invention, Compound I is contained in asolid molecular complex formed with hydroxypropyl methyl celluloseacetate succinate (HPMC-AS). As used herein, the term “solid molecularcomplex” means a composition wherein Compound I is randomly distributed(“molecularly dispersed”) within a matrix formed by HPMC-AS. In certainembodiments Compound I is present in the polymer in a final state ofsubdivision. In certain embodiments, Compound I is molecularly dispersedwithin the HPMC-AS matrix such that it is immobilized in its amorphousform. By “immobilized”, it is meant that the molecules of Compound Iinteract with molecules of HPMC-AS in such a way that they are held inthe aforementioned matrix and prevented from crystal nucleation due tolack of mobility. In some embodiments the polymer may preventintramolecular hydrogen bonding or weak dispersion forces between two ormore molecules of Compound I.

In some embodiments the ratio of the amount by weight of Compound Iwithin the solid molecular complex to the amount by weight of HPMC-AStherein is from about 1:9 to about 5:5. In an embodiment, said ratio isfrom about 2:8 to about 4:6. In another embodiment, said ratio is about3:7.

In certain embodiments of the method and kit of the present invention,the first component comprises the aforementioned solid molecular complexof Compound I and HPMC-AS blended with colloidal silicon dioxide. Incertain embodiments, the blend is at least 0.5% by weight silicondioxide. In an embodiment of the present invention, the blend is about97% complex and about 3% silicon dioxide.

In another embodiment, the first component includes a compositioncomprising the aforementioned solid molecular complex, either blended ornot blended with silicon dioxide as described above, and apharmaceutically acceptable carrier. In certain embodiments, theaforementioned complex or blend comprising the same is suspended in thecarrier. An example of a carrier is hydroxypropylcellulose (HPC). In anembodiment, the vehicle contains about 2% by weight HPC.

Each component may also contain additional agents such as preservingagents, solubilizing agents, stabilizing agents, wetting agents,emulsifying agents, sweetening agents, coloring agents, flavoringagents, salts for varying the osmotic pressure, buffers, coating agentsand antioxidants.

In certain embodiments, the first component may comprise a solidmolecular complex of Compound I and HPMC-AS blended with colloidalsilicon dioxide, hydroxypropylcellulose, Crospovidone (a disintegratingagent), magnesium stearate (a lubricant that may be used in tablet andcapsulation operations), and/or croscarmellose sodium (a disintegratingagent).

In an embodiment, the first component is a hard gelatin capsulecomprising a solid molecular complex of Compound I and HPMC-AS blendedwith colloidal silicon dioxide, hydroxypropylcellulose, magnesiumstearate, and croscarmellose sodium.

In an embodiment, the first component is a tablet comprising Compound I,or a pharmaceutically acceptable salt thereof. In an embodiment, thetablet comprises a solid molecular complex of Compound I, or apharmaceutically acceptable salt thereof, and HPMC-AS. The complex may,for example, be blended with colloidal silicon dioxide,hydroxypropylcellulose, magnesium stearate, and croscarmellose sodium.The tablet may, for example, be coated with a film coating. The filmcoating may, for example, comprise polyvinyl alcohol, titanium dioxide,polyethylene glycol 3350, talc, and iron oxide red.

In certain embodiments, the second component may comprise peginterferonalfa-2a as an injectable solution.

Pegasys® is available as an injectable solution in vials and prefilledsyringes. Each 180 μg/1.0 ml vial contains approximately 1.2 ml ofsolution to deliver 1.0 ml of drug product. Subcutaneous (sc)administration of 1.0 ml delivers 180 g of drug product (expressed asthe amount of interferon alfa-2a), 8.0 mg sodium chloride, 0.05 mgpolysorbate 80, 10.0 mg benzyl alcohol, 2.62 mg sodium acetatetrihydrate, and 0.0462 mg acetic acid. The solution is colorless tolight yellow and the pH is 6.0±0.5. Each 180 μg/0.5 ml prefilled syringecontains 0.6 ml of solution to deliver 0.5 ml of drug product.Subcutaneous (sc) administration of 0.5 ml delivers 180 μg of drugproduct (expressed as the amount of interferon alfa-2a), 4.0 mg sodiumchloride, 0.025 mg polysorbate 80, 5.0 mg benzyl alcohol, 1.3085 mgsodium acetate trihydrate, and 0.0231 mg acetic acid. The solution iscolorless to light yellow and the pH is 6.0±0.5.

Applicants have conducted studies using mice containing a human melanomaxenograft.

Applicants found that, while the combination of Compound I at 75 mg/kgbid and peginterferon alfa-2a at 900 μg 1×/wk produced significantlyincreased life span (ILS) in mice in comparison to what was achievedwith 900 μg 1×/wk peginterferon alfa-2a monotherapy, the ILS resultswere statistically equivalent to what was achieved with Compound I 75mg/kg bid monotherapy.

In order to unmask the effect of combination therapy, applicantsconducted studies in which Compound I was administered at 25 mg/kg bidand peginterferon alfa-2a was administered at 450 μg 1×/wk. In mice, 25mg/kg bid Compound I achieved 321% increased life span (ILS) and 450 μg1×/wk peginterferon alfa-2a achieved 114% ILS. By contrast, when acombined therapy of 25 mg/kg bid Compound I and 450 μg 1×/wkpeginterferon alfa-2a was administered to mice, 2843% ILS was achieved.As such, ILS achieved by the combination therapy is significantly betterthan correlative monotherapy results at p<0.05.

It is important to note that no increased toxicity was seen in thecombination groups and there was no antagonism between the two agents.

These studies indicate that treating patients with a combination ofCompound I and peginterferon alfa-2a is superior to treatment witheither agent alone, and that combining the two agents allows forreduction in the dose of either agent needed to obtain equivalent orbetter results.

EXAMPLES

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention.

Abbreviations used herein are as follows:

q.s. as much as neededx timespo orallysc subcutaneouslybid twice dailywk weekBWL body weight loss

Example 1

This example describes the formation of a suspension comprising Compound1.

A solid molecular complex comprising Compound I and hydroxypropyl methylcellulose acetate succinate (HPMC-AS) was first formed.

Compound I and HPMC-AS in a ratio of approximately 3:7, respectively,were dissolved in dimethylacetamide (DMA). The resulting solution wasthen added with stirring to very cold dilute hydrochloric acid resultingin the co-precipitation of Compound I and HPMC-AS as a solid molecularcomplex wherein Compound I was present in a nanoparticulate size range.The ratio of DMA to acid was in the range of 1:5 to 1:10.

The co-precipitate was then washed with water to remove DMA, filtered,dried to <2% moisture content and passed through a #30 mesh screen priorto evaluation. The resulting solid molecular complex was 30% by weightCompound I and 70% by weight HPMC.

The complex was then blended with colloidal silicon dioxide (availableas Aerosil® 200 from Evonik Industries AG, Essen, Germany) such that,per 100 g of the blend, 97 g was the complex and 3 g was colloidalsilicon dioxide.

An aqueous vehicle containing 2% hydroxypropylcellulose (available asKlucel® LF from Aqualon, Wilmington, Del., USA) and 1N HCL at Qs to pH4for the purpose of pH adjustment was then prepared. 23.2 mL of thevehicle was equilibrated to room temperature and slowly transferred into773.2 mg of the aforementioned blend. The resulting preparation was thenslowly mixed until a homogenous suspension was obtained. The suspensionwas stored at 2-8° C. and protected from light.

The suspension contained 9.375 mg/mL of Compound I.

Example 2

This example describes an injectable solution of peginterferon alfa-2a.

Component Amount Peginterferon alfa-2a 4.5 mg Benzyl alcohol 10.0 mgSodium chloride 8.00 mg Sodium acetate trihydrate 2.617 mg Acetic acid,glacial 0.0462 mg Polysorbate 80 0.05 mg Sodium acetate trihydrate, 10%w/v q.s. pH 6.0 Acetic acid, 10% w/v q.s., pH 6.0 Water for injectionq.s. to 1.0 mlThe solution was stored at 2 to 8° C.

Example 3

Mice were implanted with human LOX-IMVI melanoma cell xenografts. Themice, cell line used, and implantation are described below.

Female athymic Crl:NU-Foxn1nu mice were used for efficacy testing(Charles River, Wilmington, Mass., USA). Mice were 10-12 weeks of ageand weighed 23-25 grams. The health of the mice was assessed daily byobservation and analysis of blood samples taken from sentinel animals onshared shelf racks. All animals were allowed to acclimate and recoverfrom shipping-related stress for one week. Autoclaved water andirradiated food (5058-ms Pico Lab mouse chow, Purina Mills, Richmond,Ind., USA) were provided ad libitum, and the animals were kept in a 12hour light and dark cycle. Cages, bedding and water bottles wereautoclaved before use and changed weekly. All animal experiments wereconducted in accordance with the Guide for the Care and Use ofLaboratory Animals, local regulations, and protocols approved by theRoche Animal Care and Use Committee in our AAALAC accredited facility.

LOX-IMVI cells (aka LOX, National Cancer Institute—Bethesda, Md.) weregrown in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS)and 1% of 200 nM L-glutamine, scaled up, harvested, and prepared so thateach mouse received 2×10⁶ cells/0.2 ml calcium and magnesium freephosphate-buffered saline (PBS). Cells were implanted in thesubcutaneous right flank of each mouse.

Mice implanted with human xenografts were randomized into eight groupsof 10 mice each according to tumor volume so that all groups had similarstarting mean tumor volumes. The approximate starting mean tumor volumefor this study was 130 mm³.

Example 4

Compound I was formulated as a suspension as described in example 1.Peginterferon alfa-2a was formulated as an injectable solution asdescribed in example 2.

Treatment began on day 5 post-cell implant and ended at day 18 post-cellimplant. Four groups of mice developed in example 3 were used. Eachgroup was subjected to a different therapy as follows:

(1) mice receiving Compound I vehicle bid po and peginterferon alfa-2avehicle 1×/wk sc;(2) mice receiving Compound I at 75 mg/kg bid bid po;(3) mice receiving peginterferon alfa-2a at 900 μg 1×/wk sc; and(4) mice receiving Compound I at 75 mg/kg bid bid po and peginterferonalfa-2a at 900 μg 1×/wk sc.

The Compound I suspension and its corresponding vehicle were dosed usinga sterile 1 cc syringe and 18-gauge gavage needle (0.2 ml/animal) twicedaily. The peginterferon alfa-2a solution and its corresponding vehiclewere dosed using a sterile 1 cc syringe and 26-gauge needle (0.2ml/animal) once weekly on days 5 and 12 post-cell implant for a total oftwo injections.

Tumor measurements were taken once or twice per week. All animals wereindividually followed throughout the experiment.

Weight loss was graphically represented as percent change in mean groupbody weight, using the formula: ((W−W₀)/W₀)×100, where ‘W’ representsmean body weight of the treated group at a particular day, and ‘W₀’represents mean body weight of the same treated group at initiation oftreatment. Maximum weight loss was also represented using the aboveformula, and indicated the maximum percent body weight loss that wasobserved at any time during the entire experiment for a particulargroup.

Efficacy data was graphically represented as the mean tumorvolume±standard error of the mean (SEM). In addition, tumor volumes oftreated groups were presented as percentages of tumor volumes of thecontrol groups (% T/C), using the formula: 100×((T−T₀)/(C−C₀)), where Trepresented mean tumor volume of a treated group on a specific dayduring the experiment, T₀ represented mean tumor volume of the sametreated group on the first day of treatment; C represented mean tumorvolume of a control group on the specific day during the experiment, andC₀ represented mean tumor volume of the same treated group on the firstday of treatment.

Tumor volume (in cubic millimeters) was calculated using the ellipsoidformula: (D×(d²))/2, where “D” represents the large diameter of thetumor and “d” represents the small diameter.

Also, tumor regression and/or percent change in tumor volume wascalculated using the formula: ((T−T₀)/T₀)×100, where ‘T’ represents meantumor volume of the treated group at a particular day, and ‘T₀’represents mean tumor volume of the same treated group at initiation oftreatment.

Statistical analysis was determined by the rank sum test and One WayAnova and a post-hoc Bonferroni t-test (SigmaStat, version 2.0, JandelScientific, San Francisco, Calif., USA). Differences between groups wereconsidered to be significant when the probability value (p) was ≦0.05.

For survival assessment, the percent of increased life space (ILS) wascalculated as: 100×[(median survival day of treated group−mediansurvival day of control group)/median survival day of control group].Median survival was determined utilizing Kaplan Meier survival analysis.Survival in treated groups was statistically compared with the vehiclegroup and survival comparisons were done between groups using thelog-rank test (Graph Pad Prism, La Jolla, Calif., USA). Differencesbetween groups were considered significant when the probability value(p) was ≦0.05.

Toxicity

No signs of toxicity were noted in any dose group in any of the studiesdescribed as assessed by measuring changes in body weight and grossobservation of individual animals. These results are depicted in Table 1and FIG. 1.

TABLE 1 % Change in Body Weight at Max % Max % end of Study WeightWeight # animals ≧20% Group Frequency Route Day 18 Loss Gain BWLMortality Combo bid, 1x/wk po, sc 13.5 5.3 13.5 0 0 Vehicle Compound Ibid po −7.0 −7.0 −3.6 0 0 75 mg/kg Peginterferon 1x/wk sc 2.7 0.7 2.7 00 alfa-2a 900 μg Compound I bid, 1x/wk po, sc −1.1 −1.1 0.5 0 0 75mg/kg + Peginterferon alfa-2a 900 μg

Tumor Growth Inhibition (TGI)

The group receiving Compound I monotherapy at 75 mg/kg bid exhibitedgreater than 100% TGI with 10 out of 10 complete regressions (CRs). Thegroup receiving peginterferon alfa-2a monotherapy at 900 μg 1×/wkexhibited 98% TGI, 2 partial regressions (PRs) and 3 CRs out of 10. Thegroup receiving combination therapy of Compound I at 75 mg/kg bid andpeginterferon alfa-2a at 900 μg 1×/wk exhibited greater than 100% TGIwith 10 out of 10 CRs. See Tables 2 and 3 and FIG. 2.

TABLE 2 Mean Tumor Mean Tumor Volume (mm³) Volume (mm³) Start Study EndStudy Group Frequency Route DAY: 5 SEM SD DAY: 18 SD SEM Combo Vehiclebid, 1x/wk po, sc 132.83 ±2.65 ±8.38 3918.69 ±914.15 ±289.08 Compound Ibid po 134.36 ±2.52 ±7.98 0.00 ±0.00 ±0.00 75 mg/kg Peginterferon 1x/wksc 131.77 ±2.84 ±8.98 193.98 ±220.41 ±69.70 alfa-2a 900 μg Compound Ibid, 1x/wk po, sc 131.79 ±1.68 ±5.32 0.00 ±0.00 ±0.00 75 mg/kg +Peginterferon alfa-2a 900 μg

TABLE 3 % T/C % Inhibition end P value Average end of study of study Endof study % Regression per Partial Complete Animals per % Tumor GrowthGroup Day: 18 Day: 18 Day: 18 Group Regression Regression GroupInhibition Combo Vehicle — — — — 0 0 10 — Compound I −4 regression<0.001 100 0 10 10 >100 75 mg/kg bid Peginterferon alfa- 2 98 <0.001 — 23 10 98 2a 900 μg 1x/wk Compound I −3 regression <0.001 100 0 10 10 >10075 mg/kg bid + Peginterferon alfa- 2a 900 μg 1x/wk

Assessment of Survival

The group receiving Compound I monotherapy at 75 mg/kg bid exhibited3500% increased life span (ILS). The group receiving peginterferonalfa-2a monotherapy at 900 μg 1×/wk exhibited 143% ILS. The groupreceiving combination therapy of Compound I at 75 mg/kg bid andpeginterferon alfa-2a at 900 μg 1×/wk exhibited 3400% ILS. See Table 4and FIG. 3.

TABLE 4 ILS Calculations 50% 50% Treatment Vehicle % P Group Days DaysILS value Combo Vehicle — — — — Compound I 504 14 3500 <0.0001 75 mg/kgbid Peginterferon alfa-2a 34 14 143 <0.0001 900 μg 1x/wk Compound I 49014 3400 <0.0001 75 mg/kg bid + Peginterferon alfa-2a 900 μg 1x/wk

Statistical Analysis

The % TGI in the Compound I 75 mg/kg bid/peginterferon alfa-2a 900 μg1×/wk combination therapy group was statistically superior to that ofthe peginterferon alfa-2a 900 μg 1×/wk monotherapy group but equivalentto that of the Compound I 75 mg/kg bid monotherapy group. The % ILS inthe Compound I 75 mg/kg bid/peginterferon alfa-2a 900 μg 1×/wkcombination therapy group was statistically superior to that of thepeginterferon alfa-2a 900 μg 1×/wk monotherapy group but equivalent tothat of the Compound I 75 mg/kg bid monotherapy group. See Table 5.

TABLE 5 TGI ILS Treatment versus Treatment p value* p value ** CompoundI Peginterferon alfa-2a <0.05 <0.0001 75 mg/kg bid 900 μg 1x/wk CompoundI Compound I >0.05 0.1343 75 mg/kg bid 75 mg/kg bid + Peginterferonalfa-2a 900 μg 1x/wk Peginterferon Compound I <0.05 <0.0001 alfa-2a 75mg/kg bid + 900 μg 1x/wk Peginterferon alfa-2a 900 μg 1x/wk *One-WayANOVA, post-hoc Bonferroni ** Breslow-Gehan-Wilcoxon

Example 5

Compound I was formulated as a suspension as described in example 1. TheCompound I vehicle was 2.0 grams of Klucel LF in Peginterferon alfa-2awas formulated as an injectable solution as described in example 2.

Treatment began on day 6 post-cell implant and ended at day 19 post-cellimplant. Four groups of mice developed in example 3 were used. Thetreatment groups were as follows:

(1) mice receiving Compound I vehicle bid po and peginterferon alfa-2avehicle 1×/wk sc;(2) mice receiving Compound I at 25 mg/kg bid po;(3) mice receiving peginterferon alfa-2a at 450 μg 1×/wk sc; and(4) mice receiving Compound I at 25 mg/kg bid and peginterferon alfa-2aat 450 μg 1×/wk.

The Compound I suspension and its corresponding vehicle were dosed usinga sterile 1 cc syringe and 18-gauge gavage needle (0.2 ml/animal) twicedaily. The peginterferon alfa-2a solution and its corresponding vehiclewere dosed using a sterile 1 cc syringe and 26-gauge needle (0.2ml/animal) once weekly on days 6 and 13 post-cell implant for a total oftwo injections.

Tumor measurements were taken once or twice per week. All animals wereindividually followed throughout the experiment.

Weight loss was graphically represented as percent change in mean groupbody weight, using the formula: ((W−W₀)/W₀)×100, where ‘W’ representsmean body weight of the treated group at a particular day, and ‘W₀’represents mean body weight of the same treated group at initiation oftreatment. Maximum weight loss was also represented using the aboveformula, and indicated the maximum percent body weight loss that wasobserved at any time during the entire experiment for a particulargroup.

Efficacy data was graphically represented as the mean tumorvolume±standard error of the mean (SEM). In addition, tumor volumes oftreated groups were presented as percentages of tumor volumes of thecontrol groups (% T/C), using the formula: 100×((T−T₀)/(C−C₀)), where Trepresented mean tumor volume of a treated group on a specific dayduring the experiment, T₀ represented mean tumor volume of the sametreated group on the first day of treatment; C represented mean tumorvolume of a control group on the specific day during the experiment, andC₀ represented mean tumor volume of the same treated group on the firstday of treatment.

Tumor volume (in cubic millimeters) was calculated using the ellipsoidformula: (D×(d2))/2, where “D” represents the large diameter of thetumor and “d” represents the small diameter.

Also, tumor regression and/or percent change in tumor volume wascalculated using the formula: ((T−T₀)/T₀)×100, where ‘T’ represents meantumor volume of the treated group at a particular day, and ‘T₀’represents mean tumor volume of the same treated group at initiation oftreatment.

Statistical analysis was determined by the rank sum test and One WayAnova and a post-hoc Bonferroni t-test (SigmaStat, version 2.0, JandelScientific, San Francisco, Calif., USA). Differences between groups wereconsidered to be significant when the probability value (p) was <0.05.

For survival assessment, the percent of increased life space (ILS) wascalculated as: 100×[(median survival day of treated group−mediansurvival day of control group)/median survival day of control group].Median survival was determined utilizing Kaplan Meier survival analysis.Survival in treated groups was statistically compared with the vehiclegroup and survival comparisons were done between groups using thelog-rank test (Graph Pad Prism, La Jolla, Calif., USA). Differencesbetween groups were considered significant when the probability value(p) was ≦0.05.

Toxicity

No signs of toxicity were noted in any dose group in any of the studiesdescribed as assessed by measuring changes in body weight and grossobservation of individual animals. These results are depicted in Table 6and FIG. 4.

TABLE 6 % Change in Body Weight at Max % Max % end of Study WeightWeight # animals ≧20% Group Frequency Route Day 19 Loss Gain BWLMortality Combo bid, 1x/wk po, sc 15.1 3.3 15.1 0 0 Vehicle Compound Ibid po 1.3 −1.0 1.3 0 0 25 mg/kg Peginterferon 1x/wk sc 8.1 3.7 8.1 0 0alfa-2a 450 μg Compound I bid, 1x/wk po, sc 2.1 0.1 2.1 0 0 25 mg/kg +Peginterferon alfa-2a 450 μg

Tumor Growth Inhibition (TGI)

The group receiving Compound I monotherapy at 25 mg/kg bid exhibitedgreater than 100% TGI with 10 out of 10 CRs. The group receivingpeginterferon alfa-2a monotherapy at 450 μg 1×/wk exhibited greater than100% TGI, 6 PRs and 1 CR out of 10. The group receiving combinationtherapy of Compound I at 25 mg/kg bid and peginterferon alfa-2a at 450μg 1×/wk exhibited greater than 100% TGI with 10 out of 10 completeregressions (CRs). See Tables 7 and 8 and FIG. 5.

TABLE 7 Mean Tumor Mean Tumor Volume (mm³) Volume (mm³) Start Study EndStudy Group Frequency Route DAY: 6 SEM SD DAY: 19 SD SEM Combo Vehiclebid, 1x/wk po, sc 130.71 ±1.46 ±4.63 2906.48 ±463.14 ±146.46 Compound Ibid po 131.02 ±2.06 ±6.52 0.00 ±0.00 ±0.00 25 mg/kg Peginterferon 1x/wksc 131.12 ±1.19 ±3.77 122.93 ±117.57 ±37.18 alfa-2a 450 μg Compound Ibid, 1x/wk po, sc 132.29 ±1.53 ±4.84 0.00 ±0.00 ±0.00 25 mg/kg +Peginterferon alfa-2a 450 μg

TABLE 8 % T/C % Inhibition end P value Average end of study of study Endof study % Regression per Partial Complete Animals per % Tumor GrowthGroup Day: 19 Day: 19 Day: 19 Group Regression Regression GroupInhibition Combo Vehicle — — — — 0 0 10 — Compound I −5 regression<0.001 100 0 10 10 >100 25 mg/kg bid Peginterferon alfa- 0 regression<0.001 6 6 1 10 >100 2a 450 μg 1x/wk Compound I −5 regression <0.001 1000 10 10 >100 25 mg/kg bid + Peginterferon alfa- 2a 450 ug 1x/wk

Assessment of Survival

The group receiving Compound I monotherapy at 25 mg/kg bid exhibited321% increased life span (ILS). The group receiving peginterferonalfa-2a monotherapy at 450 μg 1×/wk exhibited 114% ILS. The groupreceiving combination therapy of Compound I at 25 mg/kg bid andpeginterferon alfa-2a at 450 μg 1×/wk exhibited 2843% ILS. See Table 9and FIG. 6.

TABLE 9 ILS Calculations 50% 50% Treatment Vehicle % P Group Days DaysILS value Combo Vehicle — — — — Compound I 59 14 321 <0.0001 25 mg/kgbid Peginterferon alfa-2a 30 14 114 <0.0001 450 μg 1x/wk Compound I 41214 2843 <0.0001 25 mg/kg bid + Peginterferon alfa-2a 450 μg 1x/wk

The % TGI in the Compound I 25 mg/kg bid/peginterferon alfa-2a 450 μg1×/wk combination therapy group was statistically superior to that ofthe peginterferon alfa-2a 450 μg 1×/wk monotherapy group but equivalentto that of the Compound I 25 mg/kg bid monotherapy group. The % ILS inthe Compound I 25 mg/kg bid/peginterferon alfa-2a 450 μg 1×/wkcombination therapy group was statistically superior to that eachmonotherapy group. See Table 10.

TABLE 10 TGI ILS Treatment versus Treatment p value* p value ** CompoundI Peginterferon alfa-2a <0.05 <0.0001 25 mg/kg bid 450 μg 1x/wk CompoundI Compound I >0.05 0.0464 25 mg/kg bid 125 mg/kg bid + Peginterferonalfa-2a 450 μg 1x/wk Peginterferon Compound I <0.05 <0.0001 alfa-2a 25mg/kg bid + 450 μg 1x/wk Peginterferon alfa-2a 450 μg 1x/wk *One-WayANOVA, post-hoc Bonferroni ** Breslow-Gehan-Wilcoxon

1-17. (canceled) 18: A kit comprising: (A) a first component which comprises, as an active agent, propane-1-sulfonic acid {3-[5-(4-chloro-phenyl)-1H-pyrrolo [2,3-b] pyridine-3-carbonyl]-2,4-difluoro-phenyl}-amide, or a pharmaceutically-acceptable salt thereof; and (B) a second component which comprises, as an active agent, an interferon. 19: A kit according to claim 18, for use in the treatment of a proliferative disorder. 20: A composition comprising: (A) a first component which comprises, as an active agent, propane-1-sulfonic acid {3-[5-(4-chloro-phenyl)-1H-pyrrolo [2,3-b] pyridine-3-carbonyl]-2,4-difluoro-phenyl}-amide, or a pharmaceutically-acceptable salt thereof; and (B) a second component which comprises, as an active agent, an interferon. 21: A composition according to claim 20, for use in the treatment of a proliferative disorder. 22-23. (canceled) 